Phage Selection of Cyclic Peptides Inhibiting Aminoglycoside Phosphotransferases to Control Resistant Bacteria.

The clinical threat of aminoglycoside phosphotransferases (APHs) stems from their efficient inactivation of aminoglycosides, driving multidrug resistance through broad-spectrum antibiotic modification. While peptide-based inhibitors represent a promising therapeutic modality, current candidates lack sufficient potency against APHs. To address this limitation, we employed phage display technology to screen large cyclic peptide libraries (structural diversity >1011) against APH(3')-Ia, a clinically relevant enzyme derived from Escherichia coli. Our selection identified cyclic peptide families exhibiting nanomolar binding affinities characterized by two conserved motifs: CXW(P/L)LC and CP(W/F)YC. Intriguingly, divalent cations (Mg2+ and Ca2+) enhanced peptide-APH interactions, suggesting a metal-dependent binding mechanism. Competitive fluorescence polarization assays revealed that these cyclic peptides primarily occupy the ATP-binding pocket of APH(3')-Ia, with representative candidate A-L3 demonstrating significant enzymatic inhibition. This study establishes a foundation for developing APH-targeted antibiotic adjuvants through (1) identification of novel cyclic peptide scaffolds with inhibitory potential, (2) elucidation of divalent metal ion effects on inhibitor binding, and (3) mechanistic insights into ATP-binding site competition. These findings provide critical structural and functional information to guide the rational design of next-generation antibiotic resistance breakers.